Medicament containing an effector of the glutathione metabolism together with alpha-lipoic acid for use in kidney replacement therapy

ABSTRACT

The invention relates to a medicament containing an effector of the glutathione metabolism together with α-lipoic acid, its salts and/or prodrugs as a combined preparation for the simultaneous, separate or time-controlled treatment of a defective thiol-disulfide status in kidney replacement therapy and of clinical characteristics, which indicate a disorder of the thiol-disulfide status of immunocytes. The correction of a defective thiol metabolism is of fundamental importance as a basic therapy for treating a large number of diseases of different origin, in particular however in circumstances requiring an essential kidney replacement therapy.

[0001] The invention relates to the use of the combination of n-lipoicacid and effectors of the glutathione metabolism for the treatment ofdisturbances of the cellular thiol status and accompanying disorders inkidney replacement therapy.

[0002] The fine regulation of the thiol-disulfide status is one of themost important basic requirements of biological metabolic powers. Thecentral regulation element within this system is the tripeptideglutathione, which reaches relatively high concentrations (up to 10 mM)intracellularly in reduced form. In addition to glutathione, proteinsbearing thiol groups intracellularly and in particular in cellmembrane-bound form are further important units of the thiol-disulfidestatus of each cell.

[0003] The metabolism of the disulfide cleavage and thiol groupformation regulated by various enzyme classes is in its entiretyindispensable for every normal cell function due to the variety of itsbiological functions, inter alia in cellular growth and differentiationprocesses including programmed cell death and cell protection anddetoxification mechanisms. Disturbances in this system and changes inthe concentration of thiols lead to serious cellular functionaldisorders, which only remain locally restricted in the isolated case,but as a rule adversely affect the entire organism.

[0004] It was possible to demonstrate the involvement of a disturbedthiol-disulfide status in acute and chronic disorders in a large numberof investigations.

[0005] Thus, for example, marked changes in the thiol metabolism weredetected in certain nerve cells in neurodegenerative disorders such asParkinson's disease (Brain Res Rev 1997;25:335-358). There are clearindications that as a result of this metabolic disturbance an increaseddeath of the nerve cells substantially responsible for thesymptomatology of the disorder occurs in functionally impaired areas ofthe brain, the basal ganglia (Ann Neurol 1994;36:348-355).

[0006] Decreased glutathione levels or a decreased intracellularglutathione content was furthermore found in the course of vasculardisorders and their sequelae—arteriosclerosis and cardiac infarct—in theendothelial cells lining the vascular inner wall (Med Sci Res1998;26:105-106).

[0007] Pulmonary disorders which are accompanied by a turnover of thelung tissue are regularly connected with a glutathione deficit in thetissue. In such pulmonary fibrosis, the degree of severity of thedisorder proceeds in parallel to the thiol loss (Clin Chim Acta1997;265:113-119). Severe inflammatory pulmonary disorders, investigatedin the example of adult acute respiratory distress syndrome, areaccompanied by a dysregulation of the thiol metabolism of theinflammatory cells (granulocytes) involved (Chest 1996;109:163-166).

[0008] Immunocompetent defense cells of the bronchial system (alveolarmacrophages) of smokers and patients with chronic obstructive airwaysdiseases exhibit, according to our own investigations, a severe cellularthiol deficit. The degree of the disturbance of the cellular thiolstatus in this case correlates directly with restrictions of the lungfunction (Free Radic Biol Med 2000;29:1160-1165).

[0009] In recent years, increased references to a damaged thiolmetabolism have been found in chronic kidney diseases (Ren Fail1998;20:117-124), anemia (Br J Haematol 1995;91:811-819), immaturenewborn children (Pediatr Pulmonol 1995;20:160-166), noise-relatedhearing loss (Brain Res 1998;784:82-90), inflammatory intestinaldisorders (Gut 1998;42:485-492) and in diabetes mellitus (Metabolism:Clinical and Experimental 1998;47(8):993-997).

[0010] Extensive investigations on the importance of the glutathionemetabolism in virus infections demonstrated both a relatively poorprognosis of thiol-deficient cells, based on a damaged cellular defense,and an antiviral function of the glutathione inhibiting virusreplication (Proc Natl Acad Sci USA 1997;94:1967-1972).

[0011] The cellular human immune system, consisting of, the white bloodcells granulocytes, lymphocytes and monocytes is a system reactingparticularly sensitively to a disturbance in the thiol metabolism.

[0012] Minimal changes, in particular losses of cellular glutathione,can induce a cascade-like program for the self-destruction of the cell,programmed cell death (apoptosis) (FASEB J 1998;12:479-486). Thethiol-disulfide metabolism acts here as a central control member of anintact immune system, without which the organism would not be viable.

[0013] Our own investigations showed that in particular under theconditions of a high-grade restricted kidney function and kidneyreplacement therapy necessary as a result in the form of hemodialysis orperitoneal dialysis, the cellular thiol-disulfide metabolism is severelydisturbed. This disturbance results, inter alia, in an extensive loss ofnormal cell functions, such as the phagocytic ability of peritonealmacrophages or the activatability of lymphocytes. In these patients, inaddition to the existing local immune deficit, which is characterized byfrequent infections of the abdominal cavity, a markedly decreasedimmunological defense with increased general susceptibility to infectionare regularly found. Functional disturbances and a decreasedactivatability of the lymphocytes and macrophages, and imbalances of theimmunoregulatory cytokines are in particular described here (Immunobiol1999;200:62-76).

[0014] The correction of a disturbed thiol metabolism thus attainsfundamental importance as a basic therapy in the treatment of a largenumber of disorders of different origin, but in particular under theconditions of a necessary kidney replacement therapy.

[0015] α-Lipoic acid is up to now being employed with relative successas a neuroprotective substance for the treatment of neurotoxicallyrelated paresthesias in diabetic polyneuropathy. (Diabetologica1995;38:1425-1433, Diabetes Res Clin Pract 1995;29:19-26, Diab Care1999;22:1296-1301, Drug Metab Rev 1997;29:1025-1054, DE 43 43 592 C2).The use of α-lipoic acid in further neuronal disturbances includingtinnitus and sudden deafness is moreover known from DE 44 47 599 C2 andEP 0 530 446 B1.

[0016] The cytoprotective mechanism of action is based here, in additionto the influencing of the sugar-dependent protein modification (proteinglycosylation) and a decrease in the neurotoxic ketone body genesis,finally on the antioxidative function of the α-lipoic acid and itsmetabolites (Free Radic Biol Med 1995;19:227-250).

[0017] This cell protection function was particularly investigated underthe aspect of the prevention of the oxidative turnover of essentialunsaturated fatty acids. Such an inhibition of the lipid peroxidationis, in addition to the use of α-lipoic acid as a neuroprotective, thebasis for an application as a hepatoprotective medicament in variousintoxications and liver disorders (Biochemistry 1998; 37:1357-1364).

[0018] Moreover, it was possible to show that α-lipoic acid inhibits thereplication of the HI virus at different stages of development and thuscould counteract progression of the AIDS disease. It was only possible,however, to transfer the results of these laboratory experimentsrestrictedly to clinical studies (FEBS-Lett 1996;394:9-13). The sameapplies for the demonstration of an antiinflammatory function of thesubstance for the, insulin-producing islet cells of the pancreas (AgentsActions 1993;38:60-65).

[0019] In EP 0 812 590 A2 and EP 0 427 247 B1, the use of α-lipoic acidas a cytoprotective, analgesic and as a medicament in inflammatorydiseases is disclosed.

[0020] The antioxidative properties of α-lipoic acid are based, inaddition to the ability to form chelates with metal ions and directly toeliminate radicals, in particular on the function as a strong reductant.In order to carry out this reaction intracellularly, α-lipoic aciditself must be present in reduced form, as dihydrolipoic acid. Theconversion of (disulfidic) α-lipoic acid by means of reduction to thedithiol form of dihydrolipoic acid uses, for its part, reducingequivalents, this process being catalyzed, inter alia, by the enzymeglutathione reductase (Gen Pharmacol, 1997;29:315-331). This isobviously the cause of the hitherto unsatisfactory action of thesubstance with respect to the thiol restitution.

[0021] DE 44 20 102 A1 describes a pharmaceutical combination ofα-lipoic acid and cardiovascular-active substances, in particular anorganic nitrate, a calcium antagonist, ACE inhibitor or oxyfedrine. Thepharmaceutical combination should be employed for the treatment ofcardiovascular diseases and diabetes-related diseases.

[0022] Ambroxole, i.e.trans-4-(2-amino-3,5-dibromobenzyl-amino)-cyclohexane hydrochloride isemployed as a mucolytic medicament in various administration forms inlung and bronchial diseases (WO 96 33704, GB 2239242, WO 01 05378).Moreover, the use in hyperuricemia is known from DE 35 30 761. Theaction of ambroxole as a mucolytic is based both on a stimulation of thesurfactant production of the bronchial cells and in particular on theability to eliminate free radicals (Respir Med 1998;92:609-23). Thisantioxidative activity of the substance based thereon was mainlydemonstrated on pulmonary cells (Pharmacol 1999;59: 135-141) but also inthe context of inflammatory mechanisms (Inflamm Res 1999;48:86-93).Furthermore, it is known that in vitro regulatory enzymes of theglutathione metabolism are directly influenced and peroxidativeprocesses are inhibited by the addition of ambroxole in high doses, andperoxidative processes are inhibited (Arch Vet Pol 1992;32:57-66).

[0023] Angiotensin-converting enzyme inhibitors (ACE inhibitors) areemployed with great success in the treatment of a wide range ofcardiovascular diseases.

[0024] The cause of the hypotensive action utilized here is based on theinhibition of the conversion of angiotensin I to angiotensin II.Moreover, ACE inhibitors were also described as effectors of theglutathione metabolism. In addition to investigations on relevanteffects in cardiovascular and vascular disorders (J Cardiovasc Pharmacol2000;36:503-509) general regulation principles were investigated (ClinNephrol 1997;47:243-247). The actions of ACE inhibitors bearing SHgroups, such as, for example, captopril(1-[(2S)-3-mercapto-2-methylpropionyl]-L-proline) from SH-free ACEinhibitors such as, for example, enalapril(1-{N-[(S)-1-ethoxycarbonyl-3-phenylpropyl]-L-alanyl}-L-proline) are tobe distinguished here. The former react directly as radical scavengersantioxidatively while SH-free ACE inhibitors are primarily not able todo this. Common to both groups is the influencing of the glutathioneredox cycle via the regulation of the glutathione reductase andglutathione peroxidase, and furthermore superoxide dismutase (Am JPhysiol Regulatory Integrative Comp. Physiol.2000;278:572-577).

[0025] It was therefore the object of the present invention to makeavailable novel medicaments containing thiol-reactive substances for theimproved stabilization of a damaged thiol-disulfide status, inparticular in the, case of renal insufficiency in kidney replacementtherapy, and for the restitution of the functional losses causedthereby.

[0026] This object is achieved by the medicament according to theinvention having the features of claim 1. In claim 13, the use of theactive compounds for the production of a medicament is described. Thefurther dependent claims in each case show advantageous refinements.

[0027] According to the invention, effectors of the glutathionemetabolism are in this case employed in combination with α-lipoic acid,its salts and/or its prodrugs.

[0028] Surprisingly, it was possible to show that by the administrationof the combination of α-lipoic acid and an effector of the glutathionemetabolism used according to the invention a normalization of theprimarily decreased thiol status of immune cells took place. Thethiol-stabilizing action of the combinations in this case exceeded thatof the sole use of α-lipoic acid or of the respective effectors not onlyregularly, but on the contrary superadditive effects were also able tobe detected. The restitution of the thiol status in this case coveredboth intracellular thiols and membrane-bound SH groups and is thus anexpression of a complex biological regulation. This phenomenon is basedon the fact that the effectors of the glutathione metabolism on the onehand eliminate intermediately resulting free radicals and on the otherhand increase the availability of reducing equivalents for theconversion of the α-lipoic acid from disulfidic to reduced form and thusimprove the synthesis-inducing action of the α-lipoic acid on thethiol-disulfide status.

[0029] Moreover, it was clear that a thiol-increasing action of thecombination of effectors of the glutathione metabolism and α-lipoic acidonly occurred in primarily thiol-deficient immune cells. Healthy immunecells which exhibited no alteration of the thiol-disulfide status didnot react with a further increase in the SH concentration.

[0030] The restitution of the thiol status of the immune cells wasaccompanied by a normalization of functional parameters. This related inparticular to the immunomodulatory effects in the context of theactivatability of T lymphocytes.

[0031] Furthermore, it was possible to show that the combinations usedaccording to the invention stabilized the thiol-disulfide status offurther immune cells such as the peritoneal macrophages ofdialysis-dependent patients. Before the treatment with α-lipoicacid/ambroxole or

[0032] α-lipoic acid/ACE inhibitors, the peritoneal macrophagesexhibited, in addition to a deficient thiol status, an almost completeloss of their phagocytic function and a severe disturbance of the,differentiation and cytokine synthesis, which are described as causalfor the high infection rates in these patients. These functional losseswere able to be offset by the addition of the combinations namedaccording to the invention.

[0033] This medicament is particularly suitable in kidney replacementtherapy and further syndromes in which a disturbance of thethiol-disulfide status of the immune cells occurs. In this connection,the treatment can be carried out simultaneously, in separateformulations or alternatively sequentially.

[0034] The combination preparations used according to the invention canbe administered in the customary pharmacological administration forms oras an instillate and also prophylactically and therapeutically. Theeffective dose is to be determined here in a case-related manner.Preferably, in the case of human medicinal administration to thepatient, it is between 30 and 1200 mg/d, particularly preferably between200 and 600 mg/d.

[0035] In one variant, the effector of the glutathione metabolism usedis ambroxole of the general formula I,

[0036] its salts and/or its prodrug. The dose of ambroxole for humanmedical application is in this case preferably between 7.5 and 90 mg/d,particularly preferably between 60 and 75 mg/d.

[0037] In a further variant, the effector of the glutathione metabolismused is an angiotensin-converting enzyme inhibitor (ACE inhibitor).Here, the preferred dose for human medicinal administration is between0.2 and 20 mg/d.

[0038] ACE inhibitors which can be employed here are, for example, thefollowing compounds:

[0039] A) 1-[(2S)-3-mercapto-2-methylpropionyl]-L-proline (captopril) ofthe formula II

[0040] B) 1-{N-[(S)-1-ethoxycarbonyl-3-phenylpropyl]-L-alanyl}-L-proline(enalapril) of the formula III

[0041] C)(2S,3aS,6aS)-1-{(S)-N-[(S)-1-ethoxycarbonyl-3-phenylpropyl]-alanyl}-octahydrocyclopenta[b]-pyrrole-2-carboxylicacid (ramipril) of the formula IV

[0042] The medicaments can be administered orally or alternativelyparenterally here.

[0043] Additionally, the medicaments can contain customary additives.These include, for example, aqueous solvents, stabilizers, suspending,dispersing and wetting agents.

[0044] The medicament can be prepared in any desired formulation. Theseinclude, for example, solutions, granules, powders, emulsions, tabletsand/or film-coated tablets.

[0045] According to the invention, an effector of the glutathionemetabolism is used together with α-lipoic acid, its salt and/or itsprodrugs for the production of a medicament for the treatment of adisturbance of the thiol-disulfide status of immune cells in kidneyreplacement therapy.

[0046] Likewise, an effector of the glutathione metabolism can beemployed together with α-lipoic acid, its salt and/or its prodrugs forthe production of a medicament for immunomodulatory, defense-increasingand/or anti-inflammatory treatment.

[0047] The components of the combination preparation can be present hereeither in a single formulation or in separate formulations.

[0048] The use according to the invention of the combination of α-lipoicacid and effectors of the glutathione metabolism is described in greaterdetail with the aid of the following examples and figures.

EXAMPLE 1

[0049] Influence on the Cellular Thiol Status of Peripheral Human ImmuneCells

[0050] Peripheral immune cells of healthy donors (n=9) were isolatedfrom the peripheral blood by means of density gradient centrifugation.Lymphocytes having a donor-dependent relative proportion of about 90% inthis case normally represent the main fraction of the resulting totalpopulation of mononuclear cells. 10% of the mononuclear cells arerepresented by monocytes.

[0051] The mononuclear cells obtained were taken up in special cellculture media and incubated in a gas incubator at 37° C., a relativehumidity of 98% and 5% relative air-CO₂ content. The metabolism of theprimarily resting immune cells was activated by means of mitogenicstimulation (0.5 μg/ml of phytohemagglutinin). In order to test theinfluence of the combinations used according to the invention on thethiol status of thiol-deficient immune cells, these were artificiallythiol-depleted. This was carried out by culturing in thiol-deficientmedia (RPMI 1603) according to tested procedures. Comparison culturesusing whole media (RPMI 1640) served for the definition of the bestpossible normal value under culture conditions.

[0052] The determination of the intercellular thiol content at theindividual cell level was carried out using 5-chloromethylfluoresceindiacetate (CMFDA) in flow cytofluorimetry.

[0053] Primarily nonfluorogenic CMFDA is in this case passively taken upby the cell. Binding to cytoplasmic thiol groups takes place via thechloromethyl radical. After removal of the acetate radicals bynonspecific cellular esterases, this, now cell membrane-impermeablecomplex becomes fluorogenic at an excitation wavelength λ_(ex)=490 nmwith an emission wavelength λ_(em)=520, nm. The mean fluorescenceintensity of the sample (10,000 cells) is directly proportional to theconcentration of the intracellular thiol groups.

[0054] The expression of membrane-bound thiol groups was likewisedetermined by flow cytofluorimetry. In this connection,chloromethyltetramethylrhodamine (CMTMR) was employed as a thiolconjugate under the conditions of a blocked membrane potential and of aninhibited diffusion capacity of the cells. The fluorescence intensity ofthe bound fluorochrome molecules on the cell membrane is in this case inturn proportional to the amount of the thiol groups on the cell surface.

[0055] In FIG. 1, the action of the combination of α-lipoic acid andambroxole (FIG. 1a) and α-lipoic acid and enalapril (FIG. 1b) on theintracellular thiol expression of lymphocytes is shown. The followingtable shows the results of the combination of α-lipoic acid andcaptopril. The data are shown as the ratio of the cellular fluorescenceintensity for calibration particles (beads) in each case analyzed inparallel. The active compound concentration of the respectivecombination is identical to the concentrations of the individualcomponents. intracellular thiol expression [mfi_(Beads/Ratio)] cultureα-lipoic period α-lipoic captopril acid + [d] control acid [50 μM] [10μM] captopril 0 2.88 ± 0.20 2.88 ± 0.20 2.88 ± 0.20 2.88 ± 0.20 1 2.31 ±0.20 2.81 ± 0.23 2.80 ± 0.21 2.89 ± 0.31 2 1.98 ± 0.16 2.76 ± 0.50 2.76± 0.22 2.92 ± 0.32 3 1.63 ± 0.15 2.63 ± 0.60 2.49 ± 0.26 2.88 ± 0.41 41.30 ± 0.16 2.41 ± 0.40 2.21 ± 0.36 2.91 ± 0.39 6 1.10 ± 0.13 2.23 ±0.50 1.83 ± 0.33 2.93 ± 0.35 8 0.95 ± 0.10 2.02 ± 0.30 1.02 ± 0.39 2.93± 0.41 10 0.81 ± 0.10 1.89 ± 0.30 0.91 ± 0.46 2.90 ± 0.45 12 0.69 ± 0.101.86 ± 0.68 0.76 ± 0.49 2.88 ± 0.49 14 0.65 ± 0.08 1.83 ± 0.60 0.75 ±0.56 2.86 ± 0.47

[0056] Peripheral immune cells were cultured over a period of 4 daysunder normal (control 1640) or thiol-deficient conditions (1603) for theinduction of a 10-20% strength thiol reduction. As shown in 1 a, theaddition of ambroxole in combination with α-lipoic acid beginning aftera treatment period of 48 hours resulted in a complete equalization ofthe intracellular thiol deficit. Using the combination of α-lipoic acidand the SH-free ACE inhibitor enalapril, and the SH-bearing ACEinhibitor captopril, these effects were additionally quantitativelyreinforced, and detectable in the time kinetics as early as after 24hours. Neither by α-lipoic acid alone nor by the individual applicationof the effectors was a complete equalization of the thiol deficitpossible.

[0057] The results obtained using this experimental batch for theinfluence of the combinations named according to the invention on theexpression of cell membrane-bound thiols are presented in FIG. 2 for thecombination of α-lipoic acid and ambroxole (FIG. 2a), and α-lipoic acidand enalapril (FIG. 2b); the following table shows the, results of thecombination of α-lipoic acid and captopril. Membrane-bound thiolexpression [mfi_(Beads/Ratio)] culture α-lipoic period α-lipoiccaptopril acid + [d] control acid [50 μM] [10 μM] captopril 0 2.37 ±0.45 2.37 ± 0.45 2.37 ± 0.45 2.37 ± 0.39 1 2.79 ± 0.50 2.65 ± 0.39 2.63± 0.39 2.38 ± 0.38 2 2.35 ± 0.45 2.43 ± 0.52 2.54 ± 0.41 2.42 ± 0.41 31.98 ± 0.43 2.31 ± 0.36 2.52 ± 0.38 2.49 ± 0.46 4 1.63 ± 0.43 2.26 ±0.20 2.50 ± 0.41 2.39 ± 0.52 6 1.10 ± 0.46 2.19 ± 0.13 2.46 ± 0.50 2.40± 0.50 8 0.98 ± 0.31 1.93 ± 0.20 2.01 ± 0.39 2.40 ± 0.53 10 0.96 ± 0.321.63 ± 0.16 1.68 ± 0.29 2.36 ± 0.52 12 0.95 ± 0.33 1.32 ± 0.21 1.02 ±0.51 2.38 ± 0.49 14 0.98 ± 0.33 1.34 ± 0.20 0.99 ± 0.46 2.36 ± 0.55

[0058] Under the treatment with the combination α-lipoic acid andambroxole, again, beginning after 48 hours, a significant improvement inthe membrane-bound thiol expression occurred. It was particularlyobvious here that the administration of the individual substances didnot show a significant influence at any point in time. The addition ofthe combination of α-lipoic acid and the respective ACE inhibitorsresulted both in the case of enalapril and in the case of captopril in asuperadditive effect.

Example 2

[0059] Influence on the Cellular Activation Status of Peripheral Human TLymphocytes

[0060] In the culture batch described under example 1, human Tlymphocytes were stimulated with 1.0 μg/ml of phyto-hemagglutinin.Within a culture period of 72 hours, specific, markers of the cellularactivation were demonstrated quantitatively cytofluorometrically bydetection by means of monoclonal antibodies. The influence of thecombinations used according to the invention on the activation markersCD69 (early activation antigen), CD25 (intermediate activation antigen)and CD71 (late activation antigen) of T lymphocytes was investigated. InFIG. 3, the action of the combination of α-lipoic acid and ambroxole(FIG. 3a), and α-lipoic acid and enalapril (FIG. 3b) on the activationindex of T lymphocytes is shown. Compared with normal T lymphocytes(activation index=1.0), in thiol-deficient cells a clear lowering of theactivatability, confirming the cellular functional disorder, is to benoted. After addition of α-lipoic acid, the known effect of a slightimprovement in the cellular activatability occurs, which, however, in nocase eliminates the significant difference from the normal controlgroup. Ambroxole shows no influence on one of the three activationmarkers; the ACE inhibitor enalapril is only equivalent in the case ofthe effectuation of the CD25 antigen of the α-lipoic acid. In contrast,both in the case of the combined administration of α-lipoic acid andambroxole, and of α-lipoic acid and enalapril, a raising of the T cellactivation index into the normal range was detectable. This effect wasobserved in the case of early, intermediate and late activation markers.It can thus be concluded that the normalization of the cellular thiolstatus mediated by the combined use of α-lipoic acid and the respectiveeffectors of the glutathione metabolism is accompanied by a restitutionof the cellular functionality.

Example 3

[0061] Influence on the Cellular Thiol Status of Peritoneal Macrophagesin Kidney Replacement Therapy

[0062] Peritoneal macrophages were isolated from the effluate of theperitoneal dialysis of high-grade renally insufficient patients, takenup in cell culture medium and incubated in a gas incubator at 37° C., arelative humidity of 98% and 7.5% relative air-CO₂ content. In order totest the influence of the combinations used according to the inventionon the thiol status of the peritoneal macrophages, in each case afraction was treated with α-lipoic acid, the effector of the glutathionemetabolism ambroxole or the ACE inhibitor enalapril or with thecombination of α-lipoic acid/ambroxole or α-lipoic acid/enalapril, whilein each case a further fraction was used as an untreated control.

[0063] The determination of the cellular thiol status was carried out bymeans of the measuring method described under 1. The membrane expressionof thiols was determined by means of the mean fluorescence intensity(mfi) of the sample (3000 cells/measurement) after coupling to achloromethylfluorochrome derivative.

[0064] In FIG. 4, the effect of the combination of α-lipoic acid andambroxole (FIG. 4a), and α-lipoic acid and enalapril (FIG. 4b) in thetime kinetics over 14 days is shown (n=12).

[0065] With addition of the monosubstances, in turn only an increase inthe cellular thiol expression using α-lipoic acid was to be observed,while ambroxole and the ACE inhibitor showed no effect. In contrast,with the combination of α-lipoic acid and ambroxole beginning after 72hours a clear increase in the cellular thiol expression was detectable,which after a treatment period of 4 days reached a superadditive effectand after 8 days a maximum, which exceeded the starting or control databy threefold (FIG. 4a). The combination of α-lipoic acid and an ACEinhibitor (FIG. 4b) resulted in a similar, but again markedly shortenedtime kinetics. A maximum in the superadditive action was reached here asearly as after a treatment period of 48-72 hours.

[0066] In FIG. 5, the action of the combination of α-lipoic acid andenalapril (FIG. 5b) on the membrane-bound thiol expression of peritonealmacrophages in the experimental setup described above is presented. Themembrane expression of thiols was determined by means of the meanfluorescence intensity (mfi) of the sample (3000 cells/measurement)after coupling to a chloro-methylfluorochrome derivative. In comparisonwith the results of the intracellular thiol expression, a very markedeffect of the sole administration of α-lipoic acid is to be noted here,which, however, is cancelled out again after 4 days of treatment. Incontrast to this, the combined administration of α-lipoic acid andambroxole (FIG. 5a), or α-lipoic acid and an ACE inhibitor (FIG. 5b)brings about both a primarily more marked and also superadditiveincrease, which is stable beyond the observation time period, in themembrane-bound thiol expression.

Example 4

[0067] Influence on the Phagocytic Ability of Peritoneal Macrophages

[0068] In order to make possible a characterization of the peritonealmacrophages with respect to their original functions, the phagocyticability was selected as a measurement variable.

[0069] Peritoneal macrophages were isolated analogously to the proceduredescribed under example 3 and cultured ex vivo. The determination of thephagocytic power was carried out by means of a cytofluorometric test atthe individual cell level. In this test, the macrophages were coculturedwith opsonized and fluorochrome-labelled bacteria. The amount ofbacteria taken up in a defined time period was recorded quantitativelyby means of the fluorescence intensity in the macrophages and wasregarded as a measure of their phagocytic capacity.

[0070] The influence of the combinations used according to the inventionon the phagocytic ability of the peritoneal macrophages after atreatment period of 6 days is presented in the following table.Phagocytosis rate [mfi/10,000 cells] Control 371 ± 39 α-lipoic acid [50μM] 687 ± 59 Ambroxole [10 μM] 501 ± 52 α-lipoic acid + ambroxole 1398 ±286 (p < 0.05) Enalapril [5 μM] 567 ± 59 α-lipoic acid + enalapril 1698± 241 (p < 0.05) Captopril [10 μM] 653 ± 43 α-lipoic acid + captopril1589 ± 176 (p < 0.05)

[0071] After incubation with α-lipoic acid, ambroxole or enalapril, thephagocytosis rate was increased in relation to the untreated control bythe factor 1.85 (α-lipoic acid), 1.35 (ambroxole) or 1.53 (enalapril).In contrast, it was possible using the combination of α-lipoic acid andambroxole to achieve an increase in the phagocytosis rate by the factor3.7, when using the combination of α-lipoic acid and an ACE inhibitor bythe factor 4.6 (enalapril) or 4.3 (captopril).

[0072] Moreover, it was possible to demonstrate a direct correlationbetween the phagocytosis rate and the intracellular thiol content of theperitoneal macrophages for the combination of α-lipoic acid andambroxole (r=0.79; p<0.01), α-lipoic acid and captopril (r=0.86;p<0.01), and α-lipoic acid and enalapril (r=0.82; p<0.01).

Example 5

[0073] Influence on the Degree of Differentiation and Activation and theCytokine Synthesis of Peritoneal Macrophages

[0074] Peritoneal macrophages were isolated from patients under kidneyreplacement therapy according to the processes described under example 3and cultured in the presence of the combinations of α-lipoic acid andeffectors of the glutathione metabolism named according to theinvention. After incubation for 6 days, the degree of differentiation ofthe peritoneal macrophages was determined cytofluorometrically by meansof the expression of the cell surface antigens CD15 and CD11c, and thedegree of cellular activation by means of the coexpression of theactivation antigens CD69 on CD15-positive cells and CD71 onCD11c-positive cells.

[0075] The results are summarized in the following table. CD15 CD11cCD15/69 CD11c/71 Control 1.0 1.0 1.0 1.0 α-lipoic acid 1.18 ± 0.16 1.21± 0.11 1.09 ± 0.08 1.08 ± 0.09 [50 μM] Ambroxole 0.98 ± 0.13 1.01 ± 0.090.98 ± 0.11 0.96 ± 0.1 [10 μM] α-lipoic acid + 1.29 ± 0.21 1.65 ± 0.211.49 ± 0.13 1.83 ± 0.14 ambroxole Enalapril 1.21 ± 0.22 1.23 ± 0.22 1.19± 0.12 1.10 ± 0.14 [5 μM] α-lipoic acid + 2.12 ± 0.16 1.99 ± 0.15 1.69 ±0.2 1.58 ± 0.12 enalapril (p < 0.05) (p < 0.05) Captopril 1.19 ± 0.141.26 ± 0.24 1.69 ± 0.21 1.52 ± 0.16 [10 μM] α-lipoic acid + 2.25 ± 0.22.63 ± 0.23 1.74 ± 0.19 1.61 ± 0.18 captopril (p < 0.05) (p < 0.05)

[0076] It was possible to show that the expression of the maturitymarkers CD15 and CD11c increased markedly using the combination ofα-lipoic acid and ambroxole, and significantly using the combinationα-lipoic acid and ACE inhibitor. Moreover, a marked increase in theactivation antigens CD69 and CD71 on the respective cell populations wasdetectable. The administration of the monosubstances had no influence oronly a marginal influence on the degree of differentiation andactivation of the peritoneal macrophages.

[0077] In parallel to this, in this experimental batch the cell culturesupernatants were recovered and the cytokines interleukin-6 (IL-6) andinterleukin-1 receptor antagonist (IL-1ra) contained therein,synthesized and secreted by the peritoneal macrophages, were determined.The analysis was carried out using the enzyme immunoassay technique withstandardized measuring systems and is presented in the following table,which shows the effects of α-lipoic acid and effectors of the cellularglutathione metabolism on the cytokine synthesis, of peritonealmacrophages after a treatment period of 6 days (n=10). IL-6 IL-1ra[ng/10⁶ cells] [ng/10⁶ cells] Control 53.1 ± 8.9 115.2 ± 23.4 α-lipoicacid [50 μM] 46.9 ± 6.7 119.8 ± 19.5 Ambroxole [10 μM] 51.8 ± 8.1 118.6± 21.3 α-lipoic acid + ambroxole 31.5 ± 9.2 126.8 ± 15.3 (p < 0.05) (p <0.05) Enalapril [5 μM] 41.7 ± 7.3 121.1 ± 16.9 α-lipoic acid + enalapril22.3 ± 8.1 139.8 ± 22.1 (p < 0.05) (p < 0.05) Captopril [10 μM] 42.9 ±7.7 129.4 ± 25.1 α-lipoic acid + captopril 28.1 ± 6.1 143.5 ± 18.7 (p <0.05) (p < 0.05)

[0078] In the presence of the combination of α-lipoic acid andambroxole, and the combination of α-lipoic acid and the different ACEinhibitors, a significant reduction of the IL-6 synthesis wasdetectable. This effect in turn clearly went beyond the sum of thedecrease mediated by the monosubstances. The synthesis of IL-1ra wasinduced significantly under these conditions. Here too, a superadditiveinfluence of the combination of α-lipoic acid and ambroxole or ACEinhibitors was to be noted.

Example 6

[0079] Influence on the Stability of the Thiol Restitution in PeritonealMacrophages in the Dialysis Model

[0080] The peritoneal macrophages described under example 3, which werethiol-restituted by means of the combinations used according to theinvention, were removed from this test system after 6 days and culturedin a dialysis model over a period of 14 days. For this, the peritonealmacrophages were adapted to mobile collagen IV-coated matrices andbrought into contact with conventional dialysis solution 3 times dailyfor 60 minutes each. This model served here for the induction of acombined hypoglycemic/osmotic stress. In FIG. 6, the action of thecombination of α-lipoic acid and ambroxole (FIG. 6a), and c-lipoic acidand enalapril (FIG. 6b) on the intracellular thiol expression of theperitoneal macrophages in the time kinetics is presented. The membraneexpression of thiols was determined by means of the mean fluorescenceintensity (mfi) of the sample (3000 cells/measurement) after coupling toa chloro-methylfluorochrome derivative.

[0081] While in the case of the primary thiol-restituted controls, whichwere untreated in this dialysis model, within the first 4 days an almostlinear fall of the intracellular thiol concentration was to be noted,and the combined addition of α-lipoic acid and ambroxole, and ofα-lipoic acid and enalapril, resulted in a constant intracellular thiolstatus at the level of the primary restitution. Here too, a monoeffectis detectable in particular by α-lipoic acid, which, however, is onlyshort-lasting and after about 4 days in the dialysis model shows onlyapproximately 50% of the action of the combinations.

[0082] A similar picture suggests itself in the consideration of thecourses of the membrane-bound thiol expression presented in FIG. 7.Again, the quantities obtained by the primary thiol restitution are keptconstant by use of the combination of α-lipoic acid and ambroxole (FIG.7a) or ACE inhibitors (FIG. 7b), while with addition of themonosubstances only intermediate (α-lipoic acid) or marginal effects(ambroxole, enalapril) were observed.

[0083] All in all, these experiments make it clear that theadministration of the combination of α-lipoic acid and the effectors ofthe glutathione metabolism ambroxole or ACE inhibitors stabilizes aprimarily severely damaged thiol status in different cell systems. Bymeans of this normalization, a restoration of central cellularimmunoregulatory functions moreover occurs, which is not to be notedwithout such a treatment.

1. A medicament comprising an effector of the glutathione metabolismtogether with α-lipoic acid, its salts and/or its prodrugs as acombination preparation for the simultaneous, separate or sequentialtreatment of a disturbance of the thiol-disulfide status in kidneyreplacement therapy, and syndromes in which a disturbance of thethiol-disulfide status of immune cells occurs, where as an effectorambroxole having the general formula I,

its salts and/or its prodrugs is used.
 2. The medicament as claimed inclaim 1, characterized in that the dose of α-lipoic acid, its saltsand/or its prodrugs for human medicinal administration to the patient isbetween 30 and 1200 mg/d, preferably between 200 and 600 mg/d.
 3. Themedicament as claimed in claim 1 or 2, characterized in that the dose ofambroxole, its salts and/or its prodrugs for human medicinaladministration to the patient is between 7.5 and 90 mg/d, preferablybetween 60 and 75 mg/d.
 4. The medicament as claimed in at least one ofclaims 1 to 3, characterized in that the administration is carried outorally or parenterally.
 5. The medicament as claimed in at least one ofclaims 1 to 4, characterized in that further additives are selected fromthe group consisting of aqueous solvents, stabilizers, suspending,dispersing and wetting agents.
 6. The medicament as claimed in at leastone of claims 1 to 5 in the form of a solution, granules, a powder, anemulsion, a tablet and/or film-coated tablet.
 7. The use of at least oneeffector of the glutathione metabolism together with α-lipoic acid, itssalts and/or its prodrugs for the production of a medicament for thesimultaneous, separate or sequential treatment of a disturbance of thethiol-disulfide status in kidney replacement therapy in the form of ahemo- or peritoneal dialysis process.
 8. The use of at least oneeffector of the glutathione metabolism together with α-lipoic acid, itssalts and/or its prodrugs for the production of a medicament for thesimultaneous, separate or sequential treatment of a disturbance of thethiol-disulfide status in syndromes in which a disturbance of thethiol-disulfide status of immune cells occurs, in particular forsimultaneous, separate or sequential immuno-modulatory,defense-increasing and/or anti-inflammatory treatment.
 9. The use asclaimed in claims 7 and 8, characterized in that the effector used is anangiotensin-converting enzyme inhibitor (ACE inhibitor).
 10. The use asclaimed in claim 9, characterized in that the dose of theangiotensin-converting enzyme inhibitor (ACE inhibitor) for humanmedicinal administration to the patient is between 0.2 and 20 mg/d. 11.The use as claimed in claim 9 or 10, characterized in that the ACEinhibitor used is 1-[(2S)-3-mercapto-2-methylpropionyl]-L-proline(captopril) of the formula II.


12. The use as claimed in claim 9 or 10, characterized in that the ACEinhibitor used is1-{N-[(S)-1-ethoxycarbonyl-3-phenylpropyl]-L-alanyl}-L-proline(enalapril) of the formula III.


13. The use as claimed in claim 9 or 10, characterized in that the ACEinhibitor used is (2S, 3aS,6aS)-1-{(S)-N-[(S)-1-ethoxycarbonyl-3-phenylpropyl]-alanyl}-octahydrocyclopenta[b]-pyrrole-2-carboxylicacid (ramipril) of the formula IV.


14. The use as claimed in at least one of claims 7 to 13, characterizedin that the effector of the glutathione metabolism and the α-lipoicacid, its salts and/or its prodrugs are present in a single formulation.15. The use as claimed in at least one of claims 7 to 13, characterizedin that the effector of the glutathione metabolism and the α-lipoicacid, its salts and/or its prodrugs are present in separateformulations.